Influenza viruses cause approximately 36,000 deaths annually in the United States and ~500,000 deaths worldwide per year. Strains that are extremely pathogenic have been responsible for high numbers of deaths worldwide, such as the 1918 pandemic which led to ~30 million deaths around the world. Currently, there are only two approaches available for preventing or treating epidemic and pandemic influenza, vaccination and inhibitors of viral replication. Vaccination, although highly effective against homologous strains, looses efficacy in the elderly, and it is limited by the highly mutable nature of the viru, forcing the annual reformulation of the vaccine to match the antigenicity of the current influenza virus circulating strains. A number of drugs have been approved for the treatment of influenza. These drugs inhibit virus uncoating or virus spread but the use of these relatively small number of antiviral drugs is limited by the appearance of resistant virus strains. Thus, there is a clear need for additional therapeutic modalities for the treatment of influenza virus disease. In this application, we deal with the development of a new discovery platform for the identification of chemical compounds that inhibit influenza virus RNA nuclear export. Influenza virus RNAs are imported into the host cell nucleus for replication and are then exported from the nucleus as mRNAs or vRNAs to express viral proteins or generate new viral particles, respectively. Therefore, blockage of vRNAs or viral mRNAs in the nucleus results in inhibition of virus replication. We have evidence demonstrating that we can follow single viral vRNA and mRNA segments in intact infected cell and that this process can be regulated. We will use this assay to detect inhibition of viral RNA nuclear export using our ~200,000 compound library and our high throughput imaging platform. The screen is proposed for the R21 phase of the application. For the R33 phase, the selected hits will be subjected to structure activity relationship for improving potency, selectivity, stability and toxicity profile in cell based and animal based models with various strains of influenza virus. In sum, the identified compounds by this discovery platform will likely represent a new class of potential antivirals that could be useful for antagonizing influenza virus and possibly other viruses that use a similar nuclear export machinery.